Journal: Journal of neuroinflammation

Article Title: A central role for P2X7 receptors in human microglia.

doi: 10.1186/s12974-018-1353-8

Figure Lengend Snippet: Fig. 2 Human microglia express functional P2X7Rs. a Representative tracing of a whole-cell current (holding voltage = −60 mV) activated by 5 mM ATP in a free-floating primary adult human microglia. The current displayed two distinct phases: the first (Iinitial) is the immediate effect of agonist application and the second (Ifacilitate) reflects the time-dependent facilitation. b Representative tracing of a microglial current activated by 300 μM BzATP at different holding potentials (−100 to + 40 mV). c The current-voltage (I-V) relation obtained from the peak current amplitudes of the microglia cell shown in panel b. The reversal potential is indicated by the red symbol. d Concentration-response curves for ATP and BzATP in human microglia. Symbols represent means, and vertical lines are s.e.m. of the normalized current density from 5 to 25 cells. Sigmoidal curve is the best fit obtained with a four-parameter logistic function. e Representative inhibition and recovery after a 2-min incubation in 20 μM of the P2X7R antagonist A804598. The concentration of ATP was 5 mM. f ATP-gated (5 mM) current amplitude as fraction of control #1: the current amplitude before antagonist application. Control #2 represents the IATP measured after a 2-min incubation in control ECS solution before antagonists were applied. The fact that the amplitude of control #2 is not different from that of control #1 shows that the current is fully facilitated before application of antagonist. Current is significantly inhibited by the P2X7R antagonists A438079 (50 μM, n = 15; p < 0.0001; paired t test) and A804598 (20 μM, n = 17; p < 0.0001; paired t test). The current that remains after inhibition by A804598 is unaffected by co-application of the P2X4R antagonist BX430 (5 μM, n = 10; no significant difference between A804598 alone). All antagonists were applied 2 min before ATP application

Article Snippet: A438079, A804598, BX430, and 10Panx inhibitory peptide were purchased from Tocris (Minneapolis, MN, USA).

Techniques: Functional Assay, Concentration Assay, Inhibition, Incubation, Control

Journal: Journal of neuroinflammation

Article Title: A central role for P2X7 receptors in human microglia.

doi: 10.1186/s12974-018-1353-8

Figure Lengend Snippet: Fig. 7 Stimulation of the P2X7R causes robust permeabilization of human microglia to exclusively cationic dyes. a Stimulation of the P2X7R with BzATP (300 μM) induced the uptake of the cationic dye YO-PRO-1 in human microglia. Representative fluorescence images of cells incubated for 15 min in the presence of 300 μM ATP +/−the antagonists A804598 (20 μM) or A438079 (50 μM). Each antagonist was pre-incubated for 30 min. Scale bar: 20 μm. b Quantitative comparison of YO-PRO-1 uptake by microglia. Cells were incubated for 15 min with YO-PRO-1 and 300 μM BzATP in the presence of various inhibitors: BX430 (10 μM), A438079 (50 μM), and A804598 (20 μM), n = 4 separate experiments; there was significant inhibition by P2X7R antagonists (p < 0.0001; ANOVA). c Time course of YO-PRO-1 uptake shows the average s.e.m. of fluorescence intensity over time measured from individual cells within a single field of view after application of 300 μM BzATP +/−A438079 (50 μM). d, e Activation of the P2X7R with BzATP (300 μM) does not induce the uptake of the anionic dyes Lucifer yellow (Lucifer yellow, 0.5 mM) or carboxyfluorescein (CF, 0.5 mM) in human microglia. d Example fluorescence images of cells incubated in the presence of 300 μM ATP with the anionic dyes Lucifer yellow or CF. e Quantitative comparison of anionic dye uptake by microglia. Cells were incubated for 15 min with Lucifer yellow or CF in the presence of BzATP (300 μM). There is no significant difference between dye uptake in control and treated cells (n = 4 separate experiments). f Unlike human microglia, J774A.1 mouse macrophages display strong uptake of anionic dyes. Mouse macrophages were incubated for 15 min with Lucifer yellow (0.5 mM) or CF (0.5 mM) in the presence of ATP (5 mM), n = 3 separate experiments; there was significant difference of dye RFU after ATP treatment (p < 0.0001; unpaired t test)

Article Snippet: A438079, A804598, BX430, and 10Panx inhibitory peptide were purchased from Tocris (Minneapolis, MN, USA).

Techniques: Fluorescence, Incubation, Comparison, Inhibition, Activation Assay, Control